The Impact of Blood and Seminal Plasma Zinc and Copper Concentrations On Spermogram and Hormonal Changes in Infertile Nigerian Men
The Impact of Blood and Seminal Plasma Zinc and Copper Concentrations On Spermogram and Hormonal Changes in Infertile Nigerian Men
The Impact of Blood and Seminal Plasma Zinc and Copper Concentrations On Spermogram and Hormonal Changes in Infertile Nigerian Men
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ORIGINAL RESEARCH
SUMMARY
Zinc (Zn) and copper (Cu) concentrations in sera and seminal plasma
of 60 infertile males (40 oligozoospermic and 20 azoospermic) and 40
males with evidence of fertility (normozoospermic; controls) were
estimated using atomic absorption spectrophotometry. The results
were correlated with the subject’s spermogram and hormonal levels in
order to determine their relationship and significance in male
infertility. The mean serum concentration of zinc was significantly
(p<0.01) higher in oligozoospermic males when compared to
azoospermic subjects and controls. The ratios of serum Zn to seminal
plasma Zn were 1:1, 1:3 or 1:4 in oligozoospermic, normozoospermic
or azoospermic subjects, respectively. While the mean Cu
concentration was significantly higher in serum than seminal plasma
in all groups, the Zn concentration was significantly (p<0.05) higher
in seminal plasma than serum. The Cu/Zn ratio in seminal plasma was
1
Corresponding Author: Department of Biomedical Sciences, Faculty of
Health and Wellness Sciences, Cape Peninsula University of Technology,
Bellville 7535, South Africa; e-mail: [email protected]
84 Akinloye et al
INTRODUCTION
Several trace elements have been shown to be essential for testicular
development and spermatogenesis [11, 27]. Bertrand and Vladesco [8]
first noticed the presence of zinc (Zn) in semen, which later was found
to be secreted into the seminal plasma by the prostate [13, 21].
Similarly, it has been reported that the majority of copper (Cu) present
in seminal plasma originates from the prostate. However, unlike Zn,
copper is also released by other structures of the reproductive tract
(e.g. epididymis, seminal vesicles; [34]). Seminal plasma serves as a
vehicle for spermatozoa transportation to the vagina.
Seminal plasma changes in trace element levels were related to the
fertilizing capacity of spermatozoa [25]. In human semen, Zn plays an
important role in spermatozoa physiology. Therefore, its deficiency
has been implicated in gonadal dysfunction, a decreased testicular
weight and shrinkage of the seminiferous tubules [7, 27].
Asthenozoospermia has been associated with low serum Cu
concentration, and Cu/Zn was higher in asthenozoospermic, infertile
males than in fertile males [38]. A few studies reported the possible
relationship between Cu, Zn and infertility [13, 37, 38]. These reports
varied in respect to concentrations of these elements in semen plasma
as well as their role in male infertility. This study was designed to
investigate relationships between: 1/ serum and seminal plasma
concentrations of Zn or Cu, 2/ serum or seminal plasma
concentrations of Zn or Cu and selected biophysical semen
parameters, and 3/ serum or seminal plasma concentrations of Zn or
Cu and testosterone or gonadotropin serum or seminal plasma
concentrations in infertile Nigerian men.
Zinc and copper in male infertility 85
Subjects
All voluntary males of infertile couples recruited into the study
attended infertility clinics at University College Hospital in Ibadan,
Nigeria and conformed to the specific selection criteria. The study
design included three male groups (age: 20–55 years) based on sperm
count: males with a sperm count less than 20 million/ml
(oligozoospermia; n=40; mean agerSEM: 35r1.2); males with no
spermatozoa in semen (azoospermia; n=20; mean agerSEM:
35.2r1.0); and healthy fertile control males with a sperm count greater
than 20 million/ml (normozoospermia; n=40; mean agerSEM:
36.6r1.0). Exclusion criteria were as follows: testicular varicocele,
genital infections (e.g. urethritis, prostatitis, sexually transmitted
diseases), chronic illness and serious systemic diseases (e.g. diabetes,
endocrine and metabolic disorders), heavy smoking and chronic
alcohol intake and previous groin or scrotal surgery. Moreover, male
contraceptives users (e.g. condoms, spermicides), men taking
medications for a long-term (e.g. antihypertensive drugs) and known
human immunodeficiency virus (HIV)-positive patients were excluded
from the study. The clinical evaluation and response to questionnaires
were used as the bases for subject selection. The control subjects were
recruited mainly from semen donors for intrauterine fertilization and
male partners of pregnant women and nursing mothers attending the
Antenatal Clinic of the Department of Obstetrics and Gynaecology,
University College Hospital in Ibadan. The control subjects were
recruited from a similar population, having similar demographic
characteristics. A normal semen analysis with a sperm count >20
million/ml (normozospermia) and having at least two living children
were used as criteria for selecting the control subject in addition to the
exclusion criteria used in the selection of other groups. All subjects
were required to give their informed consent. Ethical approval was
granted by the ethical committee of the College of Medicine
University of Ibadan and University College Hospital Ibadan.
86 Akinloye et al
Hormonal assays
About 10 ml of venous blood was collected from the antecubital vein
of each subject into plain tubes between 9:00 and 10:30 AM. After
clot retraction, the sample was centrifuged at 3000×g for 5 min. The
serum was collected and stored at –20°C until analyzed. After the
analysis of spermogram, the semen samples were also centrifuged at
3000×g, and supernatant (seminal plasma) was collected and stored at
–20°C until further analysis. Serum and seminal plasma LH, FSH,
prolactin (PRL) and testosterone (T) assays were carried out using an
enzyme immunoassay (EIA) developed for the special research
program in human reproduction by WHO [35]. The samples were
assayed in duplicates with acceptable values with internal variation of
less than 15%. FSH minimum detectable dose (sensitivity limit) was
0.2 IU/l, LH: 0.1 IU/l, PRL: 2.0 nmol/l and T: 0.4 nmol/l.
copper with a detection limit of 0.005 ppm for both elements. The
serum and seminal plasma metals were first released from the protein
matrix by the nitric and hydrochloric acid (1:1) wet digestion method
[15]. The samples’ elements were determined by direct aspiration of
the acidic sample into the atomic absorption spectrophotometer (AAS)
flame [28]. This complies with the specification for standardized
flame AAS quick procedure for metals when using the Buck Model
201 atomic absorption system. Similarly, the determination of
selenium (Se) and cadmium (Cd) in serum and seminal plasma of the
subjects was described previously [3, 5] with a wavelength of 196 nm
for Se and 226 nm for Cd, and detection limit of 0.15 ppm and 0.01
ppm, respectively.
Statistical analysis
The Statistical Package of Science and Social Sciences Version 15.0
(SPSS Inc.; Chicago, IL, US) software was used in statistical analysis.
Sperm biophysical characteristics were log transformed. The results
were expressed as mean±SEM. Significant differences among the
three examined groups were analyzed first by one-way ANOVA
followed by an LSD test. Significant differences between two
variables were determined by student t test. The relationships between
the examined parameters were measured with Pearson’s correlation.
RESULTS
The admission characteristics of each group of subjects were similar to
those described in our previous papers [4, 5]. The sperm biophysical
parameters of the infertile subjects were found to be significantly lower
than those of the controls, except for semen volume which was similar in
all groups. The normozoospermic group has a mean semen volume of
2.43±0.32 ml, sperm count of 72.7±5.89 million, viability of 80.5±1.35%,
percentage of normal spermatozoa 84.5±1.14%, motility 79.52±1.82% and
mean progressive motility 3.4±0.11. The oligozoospermic subjects have a
mean semen volume of 2.51±0.32 ml, sperm count 5.46±1.14 million,
viability 46.8±5.01%, percentage of normal spermatozoa 55±5.76%,
motility 35.75±4.95% and mean progressive motility 1.8±0.21.
Azoospermic males have a mean semen volume of 2.89±0.77 ml. In
addition, these males had no spermatozoa. Except for T levels, there were
no significant differences in seminal plasma hormone levels among the
three groups. Serum T was lower in normozoospermic subjects as
88 Akinloye et al
Significant differences among the three groups were analyzed by ANOVA followed
by LSD test, different superscripts depict within a row significant differences among
groups; *denote significant differences between serum and seminal plasma
concentrations of respective parameters, analyzed by Student t test.
Values are expressed as r; absolute values of hormones are available in our previous
publications [4, 5]. + positive correlation; – negative correlation; *significant
correlation p<0.05; LH: luteinizing hormone; FSH: follicle stimulating hormone;
PRL: prolactin; T: testosterone
DISCUSSION
This study is the first to report a possible contribution of serum and
seminal plasma Zn and Cu to infertility in Nigerian men. It had been
reported that Zn was high in adult testis [7] and the prostate had a
higher Zn concentration than any other organ of the body [19]. The
Zinc and copper in male infertility 91
plasma Cu level and semen volume. Also, Valsa et al. [34] concluded
that the Cu level did not correlate with the studied semen parameters.
The similar pattern in the serum-seminal plasma ratio in
normozoospermic (6:1), oligozoospermic (7:1) and azoospermic (5:1)
subjects is consistent with this conclusion. Therefore, it is not
surprising that seminal plasma Cu correlated positively with semen
volume, a parameter that is not different in the studied three groups.
The relative high serum testosterone and gonadotropins
concentrations and high seminal plasma testosterone found in infertile
subjects suggest a derangement in the mechanism for T uptake at the
cellular level in the pituitary or testes [4]. Hypergonadotropism and
hypergonadism in infertile males has been reported as an indication of
Sertoli cell failure [4]. The significant positive correlation between the
seminal plasma Zn and serum gonadotropin hormones (LH and FSH)
suggests that zinc may have a direct effect on the pituitary-gonadal-
axis. The increase in gonadotropins with resultant stimulation of
Leydig cells may be responsible for the high T level observed in our
infertile males. There is evidence that the supplementation of Zn
increased the serum T level in men and animals [9, 16,24] confirming
that a low Zn level is associated with hypogonadism [1, 11]. This may
be explained by the Zn role in activating the adenyl cyclase system,
which is involved in the stimulation of steroidogenesis [12]. Direct
stimulation of Leydig cells might enhance T production [16]. The
strong positive correlation between serum Zn and T levels supported
this claim. Although, we did not observe such an association between
the Cu level and hormones of the pituitary-gonadal-axis, the serum Cu
had a significant positive correlation with serum Zn. This implies that
Cu concentration may exert an indirect effect on the pituitary-gonadal-
axis and spermatogenesis.
We correlated our previously reported serum and seminal plasma
Se and Cd data with the data from the current study – both studies
were performed on the same population. We observed strong
relationships between the serum level of Zn or Cu and Se or seminal
plasma Cd. The serum Cu level was positively correlated with serum
Zn and Se and negatively correlated with seminal plasma Cd. We had
previously reported the importance of an optimum concentration of Se
in improving male fertility [3] and the toxic effect of Cd in infertile
males [5]. The inverse relationship between serum Zn or Cu with
seminal plasma Cd may underlie a physiological mechanism
94 Akinloye et al
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